The present invention relates to oncology and identification of oncogenes and oncoproteins useful in the diagnosis, prognosis and therapy associated with neoplasia.
Chromosome aberrations are characteristic of human cancer and include translocations, inversions, amplifications and deletions. Chromosomal translocations often generate gene fusions (i.e., fusion oncogenes) that contribute to tumorigenesis through expression of encoded oncoproteins. Some translocations fuse (i.e., physically join) promoter sequences of one gene with coding sequences of another gene leading to overexpression of wild type proto-oncoproteins. Other translocations fuse coding sequence of two genes leading to expression of chimeric oncoproteins. Chimeric fusion oncogenes/oncoproteins are specific to tumor tissue and usually to cancer type. They reproduce many aspects of cancer in animal models and are of wide interest because they define biologic pathways important in human neoplasia and are ideal targets for diagnosis and therapy.
Translocations harboring fusion oncogenes have been observed consistently in human leukemia/lymphomas and sarcomas but not in carcinomas. In fact, most chromosome abnormalities identified in carcinomas to date have consisted of deletions involving loss of growth restraining tumor suppressor genes rather than translocations involving fusion oncogenes.
The search for mechanisms underlying cancer and oncogenesis is ongoing. Understanding tumorigenesis and the reasons for uncontrolled and/or rapid cell proliferation will help researchers and clinicians develop tools for early detection, diagnosis and aggressive treatment of neoplasias.
Carcinomas are the predominant causes of cancer morbidity and death in humans. The invention relates to novel molecular markers, screening assays and therapeutic strategies for carcinoma and provides compositions and methods for diagnosing and treating carcinomas, and in some aspects particularly thyroid follicular carcinoma. The invention is premised, in part, on the discovery that PAX8 and PPARxcex3 genomic loci are able to translocate to form fusion nucleic acid molecules and polypeptides which comprise both PAX8 and PPARxcex3 sequences. Thus, the invention is based in part on the finding of a fusion oncogene designated PAX8-PPARxcex31 (or its reciprocal PPARxcex31-PAX8) in carcinoma samples. The fusion oncogene (and its reciprocal) are the result of a chromosomal translocation fusing chromosomes 2 and 3, and herein referred to as t(2;3)(q13;p25).
According to one aspect of the invention, an isolated PAX8-PPARxcex31 nucleic acid molecule is provided which comprises: (a) a nucleic acid molecule which hybridizes under stringent conditions to a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5 and SEQ ID NO:22 and which codes for a PAX8-PPARxcex31 polypeptide; (b) deletions, additions and substitutions of (a) which code for a respective PAX8-PPARxcex31 polypeptide; (c) a nucleic acid molecule that differs from the nucleic acid molecules of (a) or (b) in codon sequence due to the degeneracy of the genetic code; and (d) complements of (a), (b) or (c). The preferred PAX8-PPARxcex31 nucleic acid molecules comprise a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5 and SEQ ID NO:22. In another embodiment, the invention provides isolated nucleic acid molecules which code for a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6 and SEQ ID NO:23.
The invention provides similar aspects which relate to the reciprocal fusion, PPARxcex31-PAX8. Thus, according to one aspect of the invention, an isolated PPARxcex31-PAX8 nucleic acid molecule is provided which comprises: (a) a nucleic acid molecule which hybridizes under stringent conditions to a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO:36, SEQ ID NO:37 and SEQ ID NO:38 and which codes for a PPARxcex31-PAX8 polypeptide; (b) deletions, additions and substitutions of (a) which code for a respective PPARxcex31-PAX8 polypeptide; (c) a nucleic acid molecule that differs from the nucleic acid molecules of (a) or (b) in codon sequence due to the degeneracy of the genetic code; and (d) complements of (a), (b) or (c). The preferred PPARxcex31-PAX8 nucleic acid molecules comprise a sequence selected from the group consisting of SEQ ID NO:36, SEQ ID NO:37 and SEQ ID NO:38. In another embodiment, the invention provides isolated nucleic acid molecules which code for a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:39, SEQ ID NO:40 and SEQ ID NO:41.
According to yet another aspect of the invention, an isolated PAX8-PPARxcex31 nucleic acid molecule is provided which is selected from the group consisting of: (a) a unique fragment of a nucleic acid molecule comprising a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5 and SEQ ID NO:22 (of sufficient length to represent a sequence unique within the human genome); and (b) complements of (a), provided that the unique fragment includes a sequence of contiguous nucleotides which is not identical to any sequence selected from the group consisting of: (1) sequences having the database accession numbers of Table 1 (corresponding to each SEQ ID NO) or other previously published sequences as of the date of invention or the filing date of this application, (2) complements of (1), and optionally (3) fragments of (1) and (2).
According to yet another aspect of the invention, an isolated PPARxcex31-PAX8 nucleic acid molecule is provided which is selected from the group consisting of: (a) a unique fragment of a nucleic acid molecule comprising a sequence selected from the group consisting of SEQ ID NO:36, SEQ ID NO:37, and SEQ ID NO:38 (of sufficient length to represent a sequence unique within the human genome); and (b) complements of (a), provided that the unique fragment includes a sequence of contiguous nucleotides which is not identical to any sequence selected from the group consisting of: (1) sequences having the database accession numbers of Table 1 (corresponding to each SEQ ID NO)or other previously published sequences as of the date of invention or the filing date of this application, (2) complements of (1), and optionally (3) fragments of (1) and (2).
In one embodiment, the sequence of contiguous nucleotides is selected such that at least one, or at least two, or at least three, or at least four or more contiguous nucleotides derive from each of the source genes (i.e., PPARxcex3 or PAX8).
In one embodiment, the sequence of contiguous nucleotides is selected from the group consisting of (1) at least two contiguous nucleotides nonidentical to the sequence group, (2) at least three contiguous nucleotides nonidentical to the sequence group, (3) at least four contiguous nucleotides nonidentical to the sequence group, (4) at least six contiguous nucleotides nonidentical to the sequence group, (5) at least eight contiguous nucleotides nonidentical to the sequence group, and (6) at least ten contiguous nucleotides nonidentical to the sequence group.
In another embodiment, the unique fragment has a size selected from the group consisting of at least 8 nucleotides, at least 10 nucleotides, at least 12 nucleotides, at least 14 nucleotides, at least 16 nucleotides, at least 18 nucleotides, at least 20, nucleotides, at least 22 nucleotides, at least 24 nucleotides, at least 26 nucleotides, at least 28 nucleotides, at least 30 nucleotides, at least 40 nucleotides, at least 50 nucleotides, at least 75 nucleotides, at least 100 nucleotides, at least 200 nucleotides, at least 1000 nucleotides and every integer length therebetween as if fully cited herein.
In other embodiments, the unique fragment encodes a peptide which is a fragment of a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:23 (for unique fragments of PAX8-PPARxcex31) and SEQ ID NO:39, SEQ ID NO:40 and SEQ ID NO:41 (for unique fragments of PPARxcex31-PAX8).
According to yet another aspect, the invention provides an isolated PAX8-PPARxcex31 nucleic acid molecule comprising a sequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO:9 and SEQ ID NO:11, and an isolated PPARxcex31-PAX8 nucleic acid molecule comprising a sequence selected from the group consisting of SEQ ID NO:42, SEQ ID NO:44 and SEQ ID NO:46, a replicable vector comprising such nucleic acid molecules and a host cell comprising the replicable vector.
According to other aspects, the invention provides expression vectors, and host cells transformed or transfected with such expression vectors, comprising the nucleic acid molecules described above.
According to yet another aspect of the invention, an isolated PAX8-PPARxcex31 polypeptide is provided. The isolated PAX8-PPARxcex31 polypeptide is encoded by one or more PAX8-PPARxcex31 nucleic acid molecules of the invention. Preferably, the PAX8-PPARxcex31 polypeptide is a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6 and SEQ ID NO:23. In some embodiments, the PAX8-PPARxcex31 polypeptide comprises at least two, preferably three, and, more preferably, four or more amino acids from each of the PAX8- and PPARxcex31-derived polypeptide sequences.
According to yet a further aspect of the invention, an isolated PPARxcex31-PAX8 polypeptide is provided. The isolated PPARxcex31-PAX8 polypeptide is encoded by one or more PPARxcex31-PAX8 nucleic acid molecules of the invention. Preferably, the PPARxcex31-PAX8 polypeptide is a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:39, SEQ ID NO:40 and SEQ ID NO:41. In some embodiments, the PPARxcex31-PAX8 polypeptide comprises at least two, preferably three, and, more preferably, four or more amino acids from each of the PPARxcex31- and PAX8-derived polypeptide sequences.
In other embodiments, an isolated peptide is provided which comprises a fragment or variant of the PAX8-PPARxcex31 or PPARxcex31-PAX8 polypeptides disclosed herein, of sufficient length to represent a sequence unique within the human genome, and to identify a PAX8-PPARxcex31 or a PPARxcex31-PAX8 polypeptide, respectively. The isolated peptide may comprise at least 6, at least 8, at least 9, at least 10, at least 11, at least 12, at least 14, at least 16, at least 18 or at least 20 contiguous amino acids having a sequence of a fragment selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:23 SEQ ID NO:39, SEQ ID NO:40 and SEQ ID NO:41. The isolated peptides may also possess at least one, at least two, at least three, at least four, or more amino acids from each source polypeptide. Isolated peptides which are immunogenic are also provided. In important embodiments, the peptides comprise an amino acid sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:43, SEQ ID NO:45 and SEQ ID NO:47.
According to another aspect of the invention, a composition is provided which comprises an isolated binding agent that selectively binds to a PAX8-PPARxcex31 or PPARxcex31-PAX8 nucleic acid molecule or to a PAX8-PPARxcex31 or PPARxcex31-PAX8 polypeptide encoded by the isolated nucleic acid molecules of the invention. Such isolated binding agents include nucleic acid binding agents such as probes or primers and polypeptide binding agents such as antibodies. In one embodiment, the isolated binding agents selectively bind to a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:22, SEQ ID NO:36, SEQ ID NO:37 and SEQ ID NO:38 or a fragment thereof. In another embodiment, the isolated agent binds selectively to a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:23, SEQ ID NO:39, SEQ ID NO:40 and SEQ ID NO:41 or a fragment thereof. In important embodiments, the isolated binding agent is a peptide. In a further embodiment, the peptide is an antibody or a fragment thereof (e.g., Fab, F(ab)2, Fd and antibody fragments which include a CDR3 region which binds selectively to the PAX8-PPARxcex31 or PPARxcex31-PAX8 nucleic acid molecule or polypeptide). In even more preferred embodiments, the antibody is a humanized antibody or a chimeric antibody. The isolated binding agent may be conjugated to a detectable label. The detectable label may be selected from the group consisting of a radioactive label, an enzyme, a biotin molecule, an avidin molecule or a fluorochrome.
The isolated PAX8-PPARxcex31 and PPARxcex31-PAX8 nucleic acid molecules disclosed herein have various utilities, including their use as probes and primers as diagnostic reagents for identifying the presence of PAX8-PPARxcex31 (or reciprocal) nucleic acid molecules in biological or other samples. As an example, the isolated PAX8-PPARxcex31 nucleic acid molecules are useful in the generation of PAX8-PPARxcex31 polypeptides and PAX8-PPARxcex31 binding agents. PAX8-PPARxcex31 binding agents, in turn, can be used as reagents in diagnostic and therapeutic assays to determine the presence and/or the levels of a PAX8-PPARxcex31 nucleic acid molecule or a PAX8-PPARxcex31 polypeptide in a sample. Thus, the PAX8-PPARxcex31 nucleic acid molecules, polypeptides and binding agents of the invention can be used, inter alia, in the diagnosis or treatment of conditions characterized by the presence of a PAX8-PPARxcex31 nucleic acid molecule or a PAX8-PPARxcex31 polypeptide. In some preferred embodiments, the condition is a carcinoma. In some even more preferred embodiments, the condition is thyroid follicular carcinoma. In some embodiments, the reciprocal PPARxcex31-PAX8 nucleic acid molecules and polypeptides are also useful for detection and diagnostic assays as well as in therapeutic and screening methods of the invention.
According to another aspect of the invention, a method of identifying a tumor (e.g., follicular thyroid carcinoma) is provided. The method includes obtaining a biological sample such as a tissue or a fluid from a subject and analyzing the sample for the presence of a PAX8-PPARxcex31 or a PPARxcex31-PAX8 nucleic acid molecule or a PAX8-PPARxcex31 or a PPARxcex31-PAX8 polypeptide, or unique fragments of the foregoing nucleic acid molecules and polypeptides, wherein the presence of such a nucleic acid molecule or polypeptide identifies such a tumor.
According to still another aspect of the invention, a method of identifying the presence of a PAX8-PPARxcex31 or a PPARxcex31-PAX8 molecule in a sample is provided. The method involves analyzing the sample for the presence of a PAX8-PPARxcex31 or a PPARxcex31-PAX8 nucleic acid molecule or a PAX8-PPARxcex31 or a PPARxcex31-PAX8 polypeptide. The method may further comprise contacting the sample with at least two nucleic acid amplification primers, wherein a first nucleic acid amplification primer is capable of hybridizing to a PAX8 nucleic acid molecule and a second nucleic acid amplification primer is capable of hybridizing to a PPARxcex31 nucleic acid molecule (or if the reciprocal PPARxcex31-PAX8 fusion is to be detected, wherein a first nucleic acid amplification primer is capable of hybridizing to a PPARxcex31 nucleic acid molecule and a second nucleic acid amplification primer is capable of hybridizing to a PAX8 nucleic acid molecule); amplifying a primed nucleic acid molecule which hybridizes to the first and the second nucleic acid amplification primers; and detecting the presence of an amplified nucleic acid molecule in the sample. Preferably, the amplification product contains the fusion juncture.
According to yet another aspect of the invention, a method of identifying the presence of a PAX8-PPARxcex31 or a PPARxcex31-PAX8 nucleic acid molecule in a sample is provided. The method involves contacting the sample with at least two nucleic acid probes, wherein a first nucleic acid probe is capable of hybridizing to a PAX8 nucleic acid molecule and a second nucleic acid probe is capable of hybridizing to a PPARxcex31 nucleic acid molecule (or if the reciprocal PPARxcex31-PAX8 fusion is to be detected, wherein a first nucleic acid probe is capable of hybridizing to a PPARxcex31 nucleic acid molecule and a second nucleic acid probe is capable of hybridizing to a PAX8 nucleic acid molecule); and detecting the presence of a nucleic acid molecule in the sample which hybridizes to both the first and the second nucleic acid probes.
According to a further aspect of the invention, a method of identifying the presence of the PAX8-PPARxcex31 or a PPARxcex31-PAX8 nucleic acid molecule in a sample is provided. The method involves contacting the sample with a nucleic acid probe which is capable of hybridizing to a PAX8-PPARxcex31 or a PPARxcex31-PAX8 fusion juncture, and detecting the presence of a nucleic acid molecule in the sample which hybridizes to the nucleic acid probe. The PAX8-PPARxcex31 nucleic acid fusion juncture may comprise a sequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO:9 and SEQ ID NO:11. The PPARxcex31-PAX8 nucleic acid fusion juncture may comprise a sequence selected from the group consisting of SEQ ID NO:42, SEQ ID NO:44 and SEQ ID NO:46.
According to yet another aspect of the invention, a method of identifying the presence of PAX8-PPARxcex31 or a PPARxcex31-PAX8 polypeptide in a sample is provided. The method involves contacting the sample with at least two binding agents (e.g., antibodies), wherein a first binding agent is capable of selectively binding to a PAX8 polypeptide and a second binding agent is capable of selectively binding to a PPARxcex31 polypeptide; and detecting the presence of a PAX8-PPARxcex31 polypeptide in the sample which binds both the first and the second binding agents.
According to a further aspect of the invention, a method of identifying the presence of PAX8-PPARxcex31 or a PPARxcex31-PAX8 polypeptide in a sample is provided. The method involves contacting the sample with a binding agent (e.g., an antibody) which is capable of selectively binding to a PAX8-PPARxcex31 or a PPARxcex31-PAX8 polypeptide fusion juncture, and detecting the presence of a PAX8-PPARxcex31 or a PPARxcex31-PAX8 polypeptide in the sample which selectively binds to the binding agent.
In the foregoing embodiments, the binding agent may be an antibody or a fragment thereof.
In yet another aspect, the invention provides a method for treating a subject having a disorder characterized by the presence of a PAX8-PPARxcex31 or a PPARxcex31-PAX8 nucleic acid molecule. The method involves administering an agent to a subject in need of such treatment in an amount effective to treat the subject. In one embodiment, treating the subject refers to inhibiting the progression of the disorder. In important embodiments, the agent is a PPARxcex3 ligand and the subject is not otherwise in need of PPARxcex3 ligand treatment. The PPARxcex3 ligand may be selected from the group consisting of 5-[4-[2-(5-ethylpyridin-2-yl)ethoxyl]benzyl]thiadiazolidine-2,4-dione: (pioglitazone); 5-[4-[(1-methylcyclohexyl)methoxy]benzyl]thiadiazolidine-2,4-dione: (ciglitazone); 5-[(2-benzyl-2,3-dihydrobenzopyran)-5-ylmethyl]thiadiazoline-2,4-dione: (englitazone); 5-[(2-alkoxy-5-pyridyl)methyl]-2,4-thiazolidinedione; 5-[(substituted-3-pyridyl)methyl]-2,4-thiazolidinedione; 5-[4-(2-methyl-2-phenylpropoxy)benzyl]thiazolidine-2,4-dione; 5-[4-[3-methyoxyphenyl)-2-oxooxazolidin-5-yl]-methoxy]benzyl-2,4-thiazo-lidinedione; 5-[4-[3-(3,4-difluorophenyl)-2-oxooxazolidin-5-yl]-methoxy]benzyl-2,4-thiazo-lidinedione; 5-[4-[3-(4-chloro-2-fluorophenyl)-2-oxooxazolidin-5-yl]methoxy]benzyl-2,4-thiazolidinedione; 5-[4-[3-(4-trifluoromethoxyphenyl)-2-oxooxazolidin-5-yl]methoxy]benzyl-2,4-thiazolidinedione; 5-[4-[3-(4-trifluoromethylphenyl)-2-oxooxazolidin-5-yl]methoxy]benzyl-2,4-thiazolidinedione; 5-[4-[2-[3-(4-trifluoromethylphenyl)-2-oxooxazolidin-5-yl]ethoxy]benzyl]-2,4-thiazolidinedione; 5-[4-[2-[3-(4-chloro-2-fluorophenyl)-2-oxooxazolidin-5-yl]ethoxy]benzyl]-2,4-thiazolidinedione; 5-[4-[3-(4-pyridyl)-2-oxooxazolidin-5-yl]methoxy]-benzyl-2,4-thiazolidinedione; 5-[[4-[(3,4-dihydro-6-hydroxy-2,5,7,8-tetramethyl-2H-1-benzopyran-2-yl)methoxy]phenyl]methyl]-2,4-thiazolidinedione: (troglitazone); 4-(2-naphthylmethyl)-1,2,3,5-oxathiadiazole-2-oxide; 5-[4-[2-[N-(benzoxazol-2-yl)-N-methylamino]ethoxy]benzyl]-5-methylthiazolidine-2,4-dione; 5-[4-[2-[2,4-dioxo-5-phenylthiazolidin-3-yl)ethoxy]benzyl]thiazolidine-2,4-dione; 5-[4-[2-[N-methyl-N-(phenoxycarbonyl)amino]ethoxy]benzyl]thiazolidine-2,4-dione; 5-[4-(2-phenoxyethoxy)benzyl]thiazolidine-2,4-dione; 5-[4-[2-(4-chlorophenyl)ethylsulfonyl]benzyl]thiazolidine-2,4-dione; 5-[4-[3-(5-methyl-2-phenyloxazol-4-yl)propionyl]benzyl]thiazolidine-2,4-dione; 5-[[4-(3-hydroxy-1-methylcyclohexyl)methoxy]benzyl]thiadiazolidine-2,4-dione; 5-[4-[2-(5-methyl-2-phenyloxazol-4-yl)ethoxyl]benzyl]thiadizolidione-2,4-dione; 5-[[2-(2-naphthylmethyl)benzoxazol]-5-ylmethyl]thiadiazoline-2,4-dione; 5-[4-[2-(3-phenylureido)ethoxyl]benzyl]thiadiazoline-2,4-dione; 5-[4-[2-[N-(benzoxazol-2-yl)-N-methylamino]ethoxy]benzyl]thiadiazoline-2,4-dione; 5-[4-[3-(5-methyl-2-phenyloxazol-4-yl)propionyl]benzyl]thiadiazoline-2,4-dione; 5-[2-(5-methyl-2-phenyloxazol-4-ylmethyl)benzofuran-5-ylmethyl]-oxazolidine-2,4-dione; 5-[4-[2-[N-methyl-N-(2-pyridyl)amino]ethoxy]benzyl]thiazolidine-2,4-dione; and 5-[4-[2-[N-(benzoxazol-2-yl)-N-methylamino]ethoxy]benzyl]-oxazolidine-2,4-dione. In preferred embodiments, the PPARxcex3 ligand is troglitazone or pioglitazone.
In one embodiment, the disorder is cancer. In a preferred embodiment, the cancer is follicular carcinoma. The agent may be administered directly to a tissue. In one embodiment, the tissue is thyroid tissue.
According to another aspect of the invention, a pharmaceutical composition is provided which comprises a therapeutically effective amount of an isolated PAX8-PPARxcex31 or a PPARxcex31-PAX8 nucleic acid molecule, an isolated PAX8-PPARxcex31 or a PPARxcex31-PAX8 polypeptide, or an isolated PAX8-PPARxcex31 or a PPARxcex31-PAX8 binding agent in a pharmaceutically acceptable carrier. The pharmaceutical compositions may be useful in accordance with the therapeutic methods, including the diagnostic imaging applications, disclosed herein.
The invention also provides in another aspect a method of locating and/or visualizing cells which contain a PAX8-PPARxcex31 or a PPARxcex31-PAX8 polypeptide. The method involves contacting a binding agent which is capable of binding to the PAX8-PPARxcex31 or the PPARxcex31-PAX8 polypeptide fusion juncture and which is conjugated to a detectable label to cells containing a PAX8-PPARxcex31 or a PPARxcex31-PAX8 nucleic acid molecule or a PAX8-PPARxcex31 or a PPARxcex31-PAX8 polypeptide, and observing the locus of detectable label in the cells. In one embodiment, the contacting takes place in vivo and thus the method involves introducing (e.g., injecting) the binding agent into a subject having cells containing a PAX8-PPARxcex31 or a PPARxcex31-PAX8 nucleic acid molecule or a PAX8-PPARxcex31 or a PPARxcex31-PAX8 polypeptide, and observing the locus of detectable label in the subject. In one embodiment the binding agent is a radiolabeled antibody or radiolabeled antibody fragment which is capable of binding to the PAX8-PPARxcex31 polypeptide fusion juncture and the method involves observing the locus of radioactivity in the subject.
In another aspect, a method is provided of delivering a toxic substance to a subject having cells which contain a PAX8-PPARxcex31 or a PPARxcex31-PAX8 polypeptide. The method involves administering a toxin-conjugated binding agent which is capable of binding to a PAX8-PPARxcex31 or a PPARxcex31-PAX8 polypeptide fusion juncture to the subject.
Yet another aspect of the invention provides a method of reducing expression of an PAX8-PPARxcex31 or a PPARxcex31-PAX8 nucleic acid molecule in a cell which contains a PAX8-PPARxcex31 or a PPARxcex31-PAX8 nucleic acid molecule. In some embodiments, the invention provides a method for reducing expression of an PAX8-PPARxcex31 nucleic acid molecule in a cell which contains a PPARxcex31-PAX8 nucleic acid molecule. The method involves introducing a PAX8-PPARxcex31 or a PPARxcex31-PAX8 (or in some embodiments, a PAX8 or a PPARxcex31) antisense nucleic acid molecule into a cell, and allowing the PAX8-PPARxcex31 or the PPARxcex31-PAX8 (or the PAX8 or the PPARxcex31) antisense nucleic acid molecule to hybridize to a sense PAX8-PPARxcex31 or a sense PPARxcex31-PAX8 nucleic acid molecule thereby inhibiting expression of the sense PAX8-PPARxcex31 or the sense PPARxcex31-PAX8 nucleic acid molecule.
The invention also provides a method of inhibiting production of a PAX8-PPARxcex31 or a PPARxcex31-PAX8 polypeptide. The method involves administering to a cell which contains a PAX8-PPARxcex31 or a PPARxcex31-PAX8 nucleic acid molecule a ribozyme that cleaves a PAX8-PPARxcex31 or a PPARxcex31-PAX8 nucleic acid molecule in an amount effective to inhibit production of the PAX8-PPARxcex31 or the PPARxcex31-PAX8 polypeptide. In one embodiment, the PAX8-PPARxcex31 or the PPARxcex31-PAX8 nucleic acid molecule is a PAX8-PPARxcex31 or a PPARxcex31-PAX8 mRNA nucleic acid molecule.
In another aspect, a transgenic non-human animal is provided that has somatic and germ line cells which contain a PAX8-PPARxcex31 or a PPARxcex31-PAX8 nucleic acid molecule. The PAX8-PPARxcex31 or the PPARxcex31-PAX8 nucleic acid molecule may be selected from any of the foregoing PAX8-PPARxcex31 or PPARxcex31-PAX8 nucleic acid molecules. Expression of the PAX8-PPARxcex31 or the PPARxcex31-PAX8 nucleic acid molecule results in a transgenic non-human animal having abnormal cell growth.
In yet another aspect, the invention provides a method of screening for an agent that inhibits the production of a PAX8-PPARxcex31 or a PPARxcex31-PAX8 polypeptide. The method involves determining the level of a PAX8-PPARxcex31 or a PPARxcex31-PAX8 polypeptide in the absence of an agent, determining the level of a PAX8-PPARxcex31 or a PPARxcex31-PAX8 polypeptide in the presence of the agent, and comparing the level of a PAX8-PPARxcex31 or a PPARxcex31-PAX8 polypeptide in the presence and absence of the agent. A decrease in the level of a PAX8-PPARxcex31 or a PPARxcex31-PAX8 polypeptide in the presence of the agent is indicative of an agent that inhibits the production of a PAX8-PPARxcex31 or a PPARxcex31-PAX8 polypeptide. In one embodiment, the agent inhibits transcription of a PAX8-PPARxcex31 nucleic acid molecule. In one embodiment, the agent inhibits transcription of a PPARxcex31-PAX8 nucleic acid molecule. In another embodiment, the agent inhibits translation of a PAX8-PPARxcex31 nucleic acid molecule. In yet another embodiment, the agent inhibits translation of a PPARxcex31-PAX8 nucleic acid molecule. The method may be performed in a cell free system or in a transgenic, non-human animal.
The invention further provides, in yet another aspect, a medicament and a method of making a medicament. The medicament comprises an agent and a pharmaceutically acceptable carrier. The method involves contacting an agent in a pharmaceutically acceptable carrier. The agent may be but is not limited to a PAX8-PPARxcex31 or a PPARxcex31-PAX8 nucleic acid molecule or a fragment thereof, a PAX8-PPARxcex31 or a PPARxcex31-PAX8 antisense nucleic acid molecule, a PAX8-PPARxcex31 or a PPARxcex31-PAX8 polypeptide or fragment thereof, a PAX8-PPARxcex31 or a PPARxcex31-PAX8 binding agent, and a therapeutic agent. In certain embodiments, the agent is a PPARxcex3 ligand such as a agonist or an antagonist, provided the medicament is used, and optionally specifically formulated for use, in the treatment of disorder characterized by a PAX8-PPARxcex31 or a PPARxcex31-PAX8 nucleic acid molecule and/or polypeptide excluding uses of PPARxcex3 ligands disclosed previous to the filing date of this application or the date of invention.
In summary, the invention provides isolated PAX8-PPARxcex31 and PPARxcex31-PAX8 nucleic acid molecules, unique fragments thereof, expression vectors containing the foregoing, and host cells containing the foregoing. The invention also provides isolated PAX8-PPARxcex31 and PPARxcex31-PAX8 polypeptides, Also provided are binding agents which selectively bind such nucleic acid molecules and polypeptides, and unique fragments thereof including antibodies, and pharmaceutical compositions containing any one or more of the foregoing molecules. The compositions of the invention can be used, inter alia, in the diagnosis or treatment of conditions characterized by the presence of a PAX8-PPARxcex31 or a PPARxcex31-PAX8 nucleic acid molecule or polypeptide.
It is to be understood that all aspects and embodiments described above for PAX8-PPARxcex31equally embrace methods and compositions comprising the PPARxcex31-PAX8 nucleic acid molecule, polypeptide and unique fragments thereof.